Project Summary/Abstract Metals such as copper and iron are essential metals in biology, as their redox activities facilitate basic cellular processes ranging from antioxidant defense to respiration, along with emerging regulatory roles in cell signaling. However, this redox activity can be detrimental when their homeostasis is disrupted, leading to oxidative stress and damage associated with diseases spanning neurodegeneration, metabolic disorders, and cancer. The context in which a metal resides within a biological environment significantly influences its activity and function. Recent years have seen a rise in tools for monitoring metal ions and have illuminated the diversity in metal speciation in biology, but many of these tools are focused on probing metals in the intracellular space. The state- of-the-art methods for assessing metal status in extracellular fluids such as blood plasma focus either on absolute quantitation or evaluate a limited number of metal-containing species. While these methods have offered important insight into extreme cases of metal deficiency or overload, subtle imbalances are more challenging to diagnose and understand with the available methods. The objective of this research program is to expand and elucidate the metal speciation of the extracellular space, specifically in the blood plasma and the interstitial space. In mapping the metals in blood plasma, our strategy focuses on determining the bioinorganic chemistry of a dynamic population of biological molecules known as hormones. Hormones are the chemical messengers of the body?s endocrine system that carries information from specialized glands to target tissues and organs to maintain balance in response to internal and external stimuli. In an area that we term ?metalloendocrinology?, we seek to identify hormones that interact with metals in the plasma, determine how metals influence hormone function or how hormones may regulate the levels of metals, and assess how these interactions impact metabolism, energy consumption, daily biological rhythms, and nutritional health. Additionally, we seek to initiate investigations into the relatively-unexplored inorganic biochemistry of the interstitial space, which contains the fluid that bathes the organs. This milieu offers challenges in collection and isolation for ex vivo analysis, we thus seek to develop imaging agents targeted to this extracellular locale that respond to specific metal ions of interest. Using approaches inspired by chemical biology, biomedical engineering, and spectroscopy, we propose both bioluminescent and peptide-based imaging platforms that are coupled to chemoselective reaction-based strategies for probing interstitial metals. The insights and technologies that emerge from this proposal will directly impact nutritional treatments, hormone therapy development, and biomarker discovery for metal status.